1. Field of the Invention
Use of immunoassays for the detection of physiological compounds of interest is now widespread. Immunological reactions offer specificity and sensitivity not available in other quantitative diagnostic methodology. Despite such inherent accuracy, immunoassays are subject to a wide variety of errors. Variations in temperature and reaction times can be critical. Systems requiring separation are highly sensitive to errors in the separation of "bound" and "free" labelled species (mis-classification error). Manipulative techniques, such as the preparation, measurement and transfer of reagents (pipetting errors) are invariably important. Sophisticated assay techniques using sequential and/or non-equilibrated reactions are especially vulnerable.
The quantitation of the compound of interest in an unknown sample is determined by comparing the observed result obtained with the unknown to the result obtained when assaying several solutions of varying known concentrations (standard solutions) to obtain a standard curve. It is therefore extremely important that all the assays (both standard and unknown) be carried out in the same way and with reagents of uniform characteristics. This requires the highly accurate and reproducible transfer of reagents in each of the unknown and standard assays. Moreover, in most immunoassay systems all reactions are terminated after a precise time interval by separating the "bound" from the "free" labelled species. In order to minimize errors, such termination is usually effected after the reactions are 70-90% complete and changing slowly with time.
There is a continuing need for a simple, accurate technique for carrying out assays, in particular immunoassays, where parameters such as reaction times, amounts transferred and reacted, and the like can be accurately controlled. Moreover, there is a need to be able to perform multiple simultaneous assays where deviations as to reaction time, amount transferred, and the like can be minimized among the various unknown and standard solutions. It is particularly desirable that such techniques be easily automated to carry out assays involving multiple, sequential addition and reaction of reagents.
One problem in achieving such a technique has been the nature of the fluid being analyzed. Biological fluids, such as blood, serum and saliva, are relatively viscous and contain a high protein concentration as well as large amounts of entrapped (dissolved) gases. The transfer of such fluids often causes release of the dissolved gases which in turn causes bubbling and frothing which can interfere with the assay.
Heretofore, the transfer of such fluids in automatic assay devices has been accomplished using negative pressure, typically using a syringe or a pipette bulb, to draw the sample fluid and/or reagents into a reaction or transfer receptacle. Such negative pressure aggravates the bubbling and frothing, which in turn prevents the accurate transfer of the fluids, and in some cases interferes with the progress of the desired reaction.
The inability to freely transfer biological fluids has other adverse consequences in performing automated assays. When carrying out assays in which one of the reactants is in the solid-phase, it would be desirable to periodically determine the progress of the reaction, both to investigate the kinetics of the reaction and to accurately predict the end-point of the reaction as early as possible. However, the need to completely separate the liquid and solid phase (which potentially causes frothing in the liquid phase) to measure the label, generally precludes such a technique. Moreover, the need to wash the solid phase to remove nonspecifically bound label during the course of the assay dilutes the assay medium and affects the accuracy of the assay.
It would also be desirable to continuously or periodically agitate the biological fluid in the assay medium to accelerate the progress of the reaction. Because of the slow rate of diffusion of the large molecules involved in solution, and the limited area of immobilized reagent, assays can take many hours to reach completion. While the assay time could be reduced by agitating the reagents, the frothing induced by agitation prevents accurate performance of the assay.
A final shortcoming of the prior art has been the difficulty in preparing suitable solid-phase reagents, a process which is often time-consuming and expensive. Solid-phase reagents typically comprise test tubes (or other vessels capable of holding liquid) in which a reagent has been deposited over a portion of the inner wall of the vessel. Alternatively, polystyrene balls, glass or polysaccharide beads, and the like may be provided to support reagent in the vessels. Regardless of the exact configuration of the solid phase, it is critical that the vessels be prepared under precisely controlled conditions so that the solid phase in each vessel has the same characteristics.
2. Description of the Prior Art
Automated systems for measuring a variety of samples may be found in U.S. Pat. Nos. 3,469,438; 3,684,448; 3,723,066; and 4,087,248. A listing and review of various automated radioimmunoassay systems are provided in an article by Rogers and Miles entitled "Automation of Radioimmunoassays", RADIOIMMUNOASSAY, CRC Press, Inc. pp. 127-145 (1981).
U.S. Pat. No. 4,087,248 discloses a particular solid phase reactant comprising a transfer pipette tip having the reactant bound to its innersurface. The tip is mounted on a syringe so that reactant solutions may be drawn up by creating a negative pressure in the tip.